Stress is linked to many diseases and is thought to contribute to metastatic breast cancer (BR CA). BR CA cell attachment and migration through endothelial and basement membrane barriers are critical steps in metastasis. The effect of stress hormones on these steps are unknown. Though estrogen (ES) has been implicated in BR CA metastasis and other endothelial-related processes, e.g. atherosclerosis, the effect of ES on these steps is unknown. My broad goal is to understand the effect of stress on metastasis and to see if ES modifies the endothelial barrier to BR CA cells. Here my specific aims are to test the effects of 3 stress hormones on BR CA cell attachment and migration through an endothelial monolayer in a model system. In this model, endothelial cells will be grown over basement membrane-coated porous filters in dual-well invasion systems that permit adding BR CA cells to top wells. BR CA cell migration through the monolayer to bottom wells can be studied (Aim 1). Effects of prolactin, dexamethasone (a synthetic glucocorticoid), and norepinephrine, alone and in combinations, will be studied on endothelial and basement membrane barriers, BR CA-endothelial cell attachments, and BR CA cell migration (Aim 2). These effects will be studied in the presence and absence of ES (Aim 3). I will use biochemical assays and labelling techniques, and light, epifluorescence and electron microscopy.